Mitochondrial Fragmentation Increases with Aging in Rats

The diaphragm muscle (DIAm) is the major pump muscle for breathing. Neural control of the DIAm involves recruitment of phrenic motor neurons (PhMNs) comprising motor units that vary in their mechanical and fatigue properties. Smaller PhMNs innervating fatigue resistant DIAm fibers are highly active being recruited during breathing, while larger PhMNs are infrequently recruited during more forceful expulsive behaviors. Previously, we found that to support their higher activity levels, smaller PhMNs have higher mitochondrial volume density and are less fragmented compared to larger PhMNs. With aging, larger PhMNs are more vulnerable and ~30% are lost, and those surviving display greater mitochondrial fragmentation. We also found that older rats exhibit elevated levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFa), which in other tissue has been linked to mitochondrial fragmentation via activation of the IRE1a S724 -mediated endoplasmic reticulum (ER) stress pathway. Accordingly, we hypothesized that in 24-month-old rats, increased TNFa levels activate the pIRE1a S724 -mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally increases expression of cyclin-dependent kinases (CDK1 and CDK5) that mediate phosphorylation of DRP1 at Serine 616 (pDRP1 S616 ), and ultimately mitochondrial fragmentation. We found that serum TNFa levels increased ~3.5 fold in 24-month rats compared to 6-month rats. Consistent with elevated TNFa, we found increased pIRE1a S724 and XBP1s splicing in the cervical ventral horn of 24-month rats. In retrogradely labeled PhMNs, we found XBP1s was localized to the nucleus, consistent with transcriptional activation of gene expression. Bioinformatic analysis and ChIP assay confirmed XBP1s binding to the CDK1 and CDK5 promoter regions in rat cervical ventral horn. Both CDK1 and CDK5 mRNA and protein were elevated in the cervical ventral of 24-month rats. In aged rats, pDRP1 S616 phosphorylation increased with a corresponding increase in mitochondrial fragmentation. Our results support our hypothesis that chronic inflammation (elevated TNFa levels) in older rats activate the pIRE1a S724 -mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally targets CDK1 and CDK5 expression mediating pDRP1 S616 phosphorylation and mitochondrial fragmentation. Research Supported by NIH R01 grant AG44615. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.